With the mammalian carbonic anhydrase (CA) isozymes as models, we propose to study the selective factors which have shaped the evolution of enzymes during their ascent in humans, and the mechanisms involved in the cellular regulation of enzyme expression. These studies will focus on (1) the evolutionary origins of the human CA isozymes (CA I, CA II, and CA III), (2) the effect of mutations and evolutionary changes on their regulation and activity, and (3) the control of CA isozymes gene expression during cellular differentiation. For the coming year, we plan to continue our studies on the amino acid sequences of a number of CA isozymes purified from a variety of sources which will be used to study the comparative rates of evolution of the different CA isozymes. We will also attempt to chemically characterize 4 recently discovered variants of human red cell CA I and CA II. One of our major efforts will be to characterize not only the primary structure of the recently discovered CA III isozyme found in skeletal muscle, but by preparing antisera to CA III, we will be able to study variation in the levels of this isozyme in muscle and possible other tissues. The isolation and characterization of CA mRNA will constitute another important research goal. This mRNA will be especially useful in our studies on the molecular evolution and regulation of the CA isozymes. Finally, we will attempt to characterize CA I derived from lymphocytes cultured from individuals known to have an inherited deficiency of CA I in mature erythrocytes. This information should help us to gain insights into the basis for the inherited CA deficiency, and possibly serve as a model for other enzyme deficiencies known to result in inherited disorders.